PROJECT SUMMARY/ABSTRACT The importance of the tumor microenvironment (TME) in tumor progression, invasion, and therapy is widely recognized. Intermittent and hypoxic oxygenation (pO2) and extracellular acidosis (pHe) of tumor tissues are among the most established hallmarks in solid TME, while extracellular inorganic phosphate (Pi) has been recently identified as a new signaling molecule of importance in tumorigenesis (marker of metastatic potential). The ?v?3 and ?v?5 integrins are usually expressed at low levels in most adult epithelia but can be highly upregulated in some tumors. These integrins are involved in angiogenesis and metastasis of solid tumors, and their inhibition resulted in significant reduction of functional vessel density, retardation of tumor growth, and metastasis in vivo. In this project we propose to develop theranostic multifunctional paramagnetic trityl probes for in vivo monitoring of basic physiological parameters (pO2, pH, Pi) using electron paramagnetic resonance- based techniques and patient-derived tumor xenograft (PDX) models. Specific Aim 1: Trityl probes will be designed to have minimal toxicity, a wide range of retention times, and antiangiogenic therapeutic effects due to high affinity of the probes to the ?v?3 integrins in tumor tissue. Specific Aim 2: Functional sensitivity, specific affinity to the ?v?3 integrin in in vitro (U87MG cells), in vivo toxicity, and targeting efficacy will be studied using a U87MG xenograft mouse model. Specific Aim 3. The best probe formulations will be used to construct a real- time TME profile during tumor development and antiangiogenic treatment using trityl probes developed in Specific Aim 1 and Specific Aim 2 and cilengitide (well established anti ?v? integrin drug). We hypothesize that the TME signature can predict levels of success for ?v? integrins based on antiangiogenic therapy. We will use four different cell lines which are distinctive in invasion, metastatic, and growth rate potentials, as well as ?v?3/?v?5 integrin expression levels. We will perform measurements of Pi, pHe, and pO2 in tumors by L-band EPR. PEDRI and DCE-MRI will be used to characterize functional and spatial tumor heterogeneity. In vivo Electron paramagnetic resonance (EPR) Imaging, Proton electron double resonance imaging (PEDRI), and Dynamic contrast-enhanced (DCE)-MRI tumor characterization will be complemented with immunohistochemical, pathological, and biochemical tissue analysis. Successful completion of this proposed project will result in development of new theranostic paramagnetic multifunctional probes which can be easily modified with appropriate targeting motifs to study drug therapeutic effects.